Diagnostic tests that can be performed at the point of care of an individual, such as at the bedside of a patient, at a care provider location, or at the home of the patient, are becoming increasingly popular. The promise of such diagnostic tests is described, for example, by Leroy Hood et al., “Systems Biology and New Technologies Enable Predictive and Preventative Medicine,” Science 306, no. 5696 (Oct. 22, 2004): 640-643. Depending upon the particular diagnostic test, the substance tested may be human body fluids such as blood, serum, saliva, biological cells, urine, or other biomolecules. Diagnostic tests are not, however, limited to biomolecules since testing may be further desired on consumables such as milk, baby food, or water.
As described by Stephen F. Kingsmore, “Multiplexed Protein Measurement: Technologies and Applications of Protein and Antibody Arrays,” Nature Reviews, Drug Discovery 5, no. 4 (April 2006), pages 310-320, and Robert F. Service, “PROTEOMICS: Proteomics Ponders Prime Time,” Science 321, no. 5897 (Sep. 26, 2008): 1758-1761, multiplexed measurement platforms such as protein arrays are a promising diagnostic technology that are currently being explored in conducting the diagnostic tests described above. Such multiplexed measurement platforms frequently incorporate affinity based sensors which are considered to be the state-of-the-art in detection of biomarkers.
Affinity based sensors function according to a “key-lock” principal in which a molecule with very high association factor to the marker of interest is used for detection. For example, a pregnancy test kit may incorporate a monoclonal antibody specific to a β-subunit of hCG (βhCG). The antibody is conjugated with a tag, e.g., gold, latex, or fluorophore, which is used for detection. If the targeted molecule binds with the conjugated antibody, the tagged key-lock pair will be detectable such as by a visible test line.
ELISA plates and microarrays (e.g., Nucleic Acid, peptide, and protein) incorporate a similar principal. FIG. 1 depicts an ELISA assay 10 wherein antibodies 12 are immobilized on a substrate 14. The substrate 14 may be positioned within a well (not shown). A blocker 16 is provided to cover the surface of the substrate around the antibody 12. In a typical ELISA assay, a sample 18 is then added to the well in which the primary antibody 12 is immobilized. Next, the sample is incubated for some time. During incubation, the blocker 16 prevents the molecules of interest in the sample from binding to the surface of the substrate 14 in order to avoid false binding. During incubation, some of the molecules of interest 18 become bound with some of the antibodies 12 as depicted in FIG. 2. After incubation, the remaining sample is washed to remove the unbound primary antibodies 18.
Subsequently, a secondary antibody 20 with a bound label 22 is added to the well, incubated, and washed resulting in the configuration of FIG. 3. As depicted in FIG. 3, the labeled secondary antibodies 20 are bound to the molecules of interest 18 that are in turn bound to the antibodies 12. Accordingly, the number of labels 22 bound by the antibodies 20 to the antigen 18 is proportional to the concentration of the target antigen. Depending on the label used, the number of labels can be finally detected using colorimetry, amperometry, magnetometry, voltammetry, luminescence, or fluorescence detection. Other label-free antibody processes such as surface plasmon resonance may alternatively be used.
Various issues arise when incorporating an affinity based multiplexed biomolecule detection platform in conducting tests including bias and variation. These issues are detailed by Philipp Angenendt, “Progress in Protein and Antibody Microarray Technology,” Drug Discovery Today 10, no. 7 (Apr. 1, 2005), pages 503-511, and Paul K. Tan, et al., “Evaluation of Gene Expression Measurements from Commercial Microarray Platforms,” Nucleic Acids Research 31, no. 19 (Oct. 1, 2003), pages 5676-5684. In summary, molecules immobilized on a substrate can denature and lose their binding capacity. The extent of such degradation varies depending upon the immobilized molecule and the type and conditions of immobilization. Consequently, some areas of a particular microarray may always express or not express regardless of whether or not a molecule of interest is present in a sample.
Various approaches to mitigate the errors encountered when using multiplexed measurement platforms have been developed including the provision of a coefficient of variation indicative of the consistency between various test sites on a platform or the consistency between various platforms. A coefficient of variation, however, provides only an indication of the consistency between test sites or platforms. A coefficient of variation does not account for inconsistencies between samples such as the presence of interfering molecules. Moreover, performing control experiments to identify interfering molecules or other variations in the sample or processing of a multiplexed measurement platform are prohibitive in many applications.
Another issue that arises in the development of diagnostic testing devices is the complexity of associations in gene/biomarker discovery. These complex associations may lead to unexpected variations in biological assays as reported by David a Lacher, et al., “Estimate of Biological Variation of Laboratory Analytes Based in the Third national Health and Nutrition Examination Survey,” Clinical Chemistry, 51, no. 2 (Feb. 1, 2005), pages 450-452, And Alan Aderem, “Systems Biology: Its Practice and Challenges,” Cell 121, no. 4 (May 20, 2005), pages 511-513.
The complexity of the associations in gene/biomarker discovery makes development of readily understandable depictions of the associations, such as creation of pathways, heat maps, interaction networks, etc., problematic. Moreover, the manner in which a molecule associates varies according to different test environments. One color coded approach to depicting an interaction network is described by Richard B. Jones, et al., “A Quantitative Protein Interaction Network for the ErbB Receptors Using Protein Microarrays, Nature 439, no. 7073 (Jan. 12, 2006), pages 168-174. Development of the color coded depiction, however, was the result of extensive effort and is thus generally prohibitive.
Accordingly, a need exists for a device and method of providing a confidence test for an assay. A further need exists for providing a quality metric for assays such as multiplexed assays, e.g., protein arrays, competitive assays, or bead based arrays, as well as low cost devices, e.g., lateral flow devices, or other biochips. A device and method which provides a quality metric without the need of a control sample unique to the array would be beneficial. A device and method which provides a quantitative evaluation of gene/biomarker associations under different test conditions would be further beneficial.